SPECTROPHOTOMETRIC STUDIES ABOUT AMYLASE ACTIVITY IN STARCH HYDROLYSIS REACTION INTERNAL REPORT 2012 CĂRĂBAN ALINA, POPA VASILICA, MACOCIAN EUGEN, FILIP SANDA University of Oradea, Romania Abstract The experimental work is aimed to determine the influence of some factors such as cations and anions, the contact surface between starch and amylase and the amylase extraction depending on the degree of damage to the endosperm using the DNS method in order to determine the amylase activity in starch hydrolysis. I. INTRODUCTION The amylase activity can be measured following the decrease of the viscosity of a starch solution, the decrease of the turbidity of a starch suspension, the decrease of the intensity of a starch-iodine reaction and the increase of the reducing groups in the reaction medium. The last method is in agreement with the EC-IUB demands. The amylase activity is measured using a colorimetric method with DNS reagent (3,5-dinitrosalicylic acid) after Hosttettler and co., modified by the authors in
order to ensure the appropriate conditions for the hydrolyse of starch in baking industry; - The hydrolyse reaction had place at ph 5,5 (the optimum ph for wheat amylase); - As enzymatic activator we used a solution of CaCl 2 (Ca ions are activators for the wheat amylase) in appropriate concentration 0,01M; - The hydrolyse reaction occurred at 30ºC, in accordance with that of the dough fermentation in baking industry. The starch is hydrolysed on the catalytic action of amylase to fragments, which can be determined with 3,5-dinitrosalicylic acid, due to their semiacetalic reducer groups. The formed nitroaminosalycilic acid concentration is corresponded to the enzymatic activity of amylase. II. EXPERIMENTAL Reagents Soluble starch supplied by Merck, Darmstadt was used in 1% concentration in acetate buffer solution at ph 5,5 which contain CaCl 2 0,1M. The DNS reagent (Merck, Darmstadt) was obtained from 1g 3,5-dinitrosalcylic acid dissolved in 20 ml NaOH 2N, adding 30g double tartrat of natrium and potassium and completed with distillate water to obtain 100ml solution. It was used an enzymatic extract of alpha and beta amylase from wheat flour prepared by extracting 10 g wheat flour in 100 ml distillate water for 30 minute using a magnetic stirring, than centrifuging at 6000 rpm and the supernatant obtain was dilute 10 times in distillate water. Maltose was used as standard solution in the concentration as 100 g/ml. Starch was used as enzyme substrate in the following forms: soluble starch
A1 - Merck soluble starch (1% soluble starch dissolved in phosphate buffer ph 7, containing 0.01 M CaCl 2 ) in order to determine the optimum ph of action of α - and β - amylase in wheat. A2 - Merck soluble starch 1% in acetate buffer ph 5.5, used to determine the influence of the presence of cations and anions in the hydrolysis reaction and the determination of optimal concentration of calcium ions. A3 - Merck soluble starch 1% in ph 5.5 buffer containing 0.01 M CaCl2 in order to determine the optimum temperature of amylase activity in the hydrolysis reaction; granular starch A4 - granular starch has been obtained from the washings of gluten, which were collected and centrifuged consecutively for 5 minutes at 6000 r/min and successive washes, after which it was dried at room temperature for 72 hours. Granular starch thus obtained (A4) was subjected to a grinding operations laboratory mill for 5 minutes, 10 minutes, 20 minutes and 25 minutes, achieving granular starch A5, A6, A7 and A8, with varying degrees damage to grain starch. The damage degree of the starch granule was revealed under a microscope by staining with iodine. Colorimetric analysis The reaction mixture formed by soluble starch solution, the amylase extract and the analysed factor was incubated for 5 minutes at 30ºC for the enzymatic hydrolyse reaction and then the reaction was stopped with DNS reagent and by boiling the reaction medium for 5 minutes. After cooling, the mixture was colorimeter at 546 nm related to distillate water.
Optical density Two countries, one goal, joint success To determine the reducing sugars existing in the reaction medium at initial moment, for all samples were made controls, identically with the tests, except that in the controls there was no enzyme. To transform the optical densities read for the tests and controls in moles maltose it was made a standard curve in concordance with the data from table 1. The experiment results were calculated as follow: the optical densities for the controls were subtracted from the test extinction and the results were expressed in moles maltose enzymatic delivered/1ml/1min. Table no. 1. Standard curve Maltose (ml) 0,1 0,3 0,5 0,7 1 - Distillate water (ml) 0,9 0,7 0,5 0,3-1 DNS reagent (ml) 1 1 1 1 1 1 Boiling 5 minutes Distillate water (ml) 8 8 8 8 8 8 Extinction 0,150 0,305 0,455 0,605 0,765 0,800 1,0 0,8 Equation y = a + b* Adj. R-Square 0,99126 Value Standard Error optical density Intercept 0,09701 0,01964 optical density Slope 0,69037 0,03238 0,6 0,4 0,2 0,0 0,0 0,2 0,4 0,6 0,8 1,0 1,2 Maltose (microgram/ml) Figure 1. The standard curve
III. RESULTS AND DISSCUSIONS 1. The influence of some cations over the amylase activity in starch hydrolysis reaction We analyzed the enzymatic activity of and - amylase from wheat in the presence of Ca 2+, Ba 2+, Sr 2+, Ni 2+, K +, Li +, Al 3+, Sb 3+, Fe 3+, Cr 3+, Mn 2+, Hg 2+, NH + 4, Zn 2+, Co 2+, Cd 2+ ions using colorimetric method for determination of amylase activity with DNS reagent, using as substrate soluble starch A2 and as amylase an aqueous extract E1. Work Mode No.1 Blank Starch (ml) A2 0,5 0,5 DW (ml) 0,3 0,3 Cation (ml) - 0,1 Enzyme (ml) E1 0,1 0,1 Incubation 5 minute at 30 0 C DNS (ml) 1 1 Boiling 5 minutes DW (ml) 8 8 From practical results obtained is noted that the calcium ion is the only cation activator of amylase activity, other cations studied had different effects as amylase inhibitory activity [3]. Inhibitory action is embodied in five directions: 1. ions K + and Al 3+ have a weak inhibitory effect; 2. ions Ba 2 +, Sr 2+, Li +, Ni 2+ have a moderate inhibitory effect; 3. ions Zn 2 + and Co 2+ are stronger inhibitors; 4. ions NH + 4, Fe 3+, Cr 3+, are powerful inhibitors; 5. ions Mn 2+, Hg 2+, Cd 2+ denaturated amylases.
Amylase is an enzyme requires the presence of metal ions of calcium in its molecule for its enzymatic activity. Calcium is bound by ionic bonds with carboxyl groups in the structure of amylase, amylase molecule so that each atom contains closely related gram of Ca2 + / molecule. As such, the calcium ion is an activator of amylase activity [4]. Other cations studied the different effects of inhibition of amylase activity by altering the conformation space of amylase in varying degrees, to distortion and precipitation reactions with metals present in the hydrolysis of starch molecules and destabilize the attraction that the groups OH of starch. The influence of the presence of metal ions in the hydrolysis of starch with amylase in wheat flour is shown in Figure 2. Figure 2. The influence of some cations on the amylase activity 2. The influence of the presence of some anions in the starch hydrolysis with amylases It was studied using colorimetric method for determining amylase activity in DNS the influence of the presence of the following anions HCO 3, CO 3 2, HSO 4,
SO 2 4, NO 2, NO 3, HCOO, C 6 H 5 O 3 7, H 2 PO 4, HPO 2 4, Cl, CH 3 COO, in the hydrolysis of starch by amylase of wheat flour using as substrate, soluble starch A2 and the enzyme amylase aqueous extract E1 In parallel, a blank test was performed and containing no anion. Table no.3 Work Mode no. 3 Blank s Starch A2(ml) 0,5 0,5 DW (ml) 0,3 0,3 Anion (ml) - 0,1 Enzyme E1(ml) 0,1 0,1 Incubation 5 minutes at 30 0 C DNS (ml) 1 1 Boiling 5 minutes DW (ml) 8 8 Figure 4. The influence of anions on the amylase activity
From the results obtained, it appears that carbonate and bicarbonate ions are moderate inhibitors for amylase activity. The other anions have no influence over the amylase activity. Therefore the use of bicarbonate ion as additional chemical for fermented dough causes a decrease of hydrolytic activity of amylase, an aspect that can be used as a way of correcting the grain quality with high amylase activity (wheat grass, wheat being attacked by bedbugs wheat and so on). hydrolysis 3. The influence of amylase concentration over the granular starch It was studied the influence of amylase concentration over the hydrolysis of starch granules using the colorimetric method for the determination of amylase activity with DNS reagent. It was used as enzyme substrate A6 granular starch and the enzyme amylase aqueous extract of wheat flour E1 in different concentrations. From the experimental data obtained, it is observed that with increasing enzyme concentration ("dispersion" lower enzyme) occurs an increase in enzyme activity. This indicates that not all molecules present in the reaction medium amylase is active, only those who come in direct contact with the starch granule. Granular starch is deposited on the bottom so that the area of action of amylase is limited. If the number of amylase molecules in contact with granular starch decreases due to the increase in amylase release molecules (low concentration of amylase solution), the amount of hydrolysis products decreased. The experimental results are plotted in Figure 5.
Work Mode no. 4 1 2 3 4 5 Starch A6 (g) 1 1 1 1 1 1 DW (ml) 30 25 20 15 10 5 Enzyme (ml) 5 5 5 5 5 5 Incubation 15 minutes at 30 0 C Prelevation (ml) 5 5 5 5 5 5 DNS (ml) 1 1 1 1 1 1 Boiling 5 minutes DW (ml) 4 4 4 4 4 4 6 Figure 5. The influence of amylase concentration over hydrolysis of starch 4. The determination of the contact surface influence between starch granules and amylase We observed the influence of surface contact between starch granules and amylase extract using the working method, colorimetric determination of amylase activity DNS reagent. We used samples of granular starch A6 1% as E1 enzyme
substrate and the enzyme amylase extract the same levels and working conditions, but placed in beakers of different volumes (based on the different surfaces). Work Mode no. 5 1 2 3 Starch A6 (g) 1 1 1 DW (ml) 10 10 10 Enzyme (ml) 5 5 5 Incubation 15 minutes In each sample was taken 5 ml of hydrolysis products over which added 1 ml reagent DNS, was incubated at boil 5 minutes and then the samples were diluted with 4 ml of distilled water after that were colorimetry at 546 nm. The influence of the contact surface granular starch - amylase on hydrolysis is illustrated in Figure 6. Figure 6. Influence of the contact surface over granular starch hydrolysis with amylase It is noted that for a larger contact surface, amylase activity increases. Starch granules, accumulating at the bottom of the reaction vessel, limiting access only amylase molecules floor of granular starch - amylase solution so, how this area is
higher, the more complex starches - active amylase formats is higher and therefore the hydrolysis products formed in greater quantity. 5. Determination the influence of substrate condition over amylase activity It was determined the influence of substrate condition on amylase activity in wheat flour using colorimetric method for the determination of amylase activity DNS reagent as No.6 mode. It was used a biochemical sample containing the enzyme substrate granular starch A6 (compared to a control performed with Merck soluble starch A3), and the aqueous extract of amylase enzyme E 1. From practical results it is observed that soluble starch hydrolysis go much faster than that of granular starch (about 4 times faster). Granular starch is more resistant to hydrolysis than the soluble starch. Work Mode No.6 Blank Starch (g) Starch (A3) 0,5 Starch (A 6 ) 0,5 DW (ml) 20 20 Enzyme E1 (ml) 5 5 Incubation 15 minutes at 30 0 C Prelevation (ml) 5 5 DNS (ml) 1 1 Boiling 5 minutes DW (ml) 4 4 The influence of substrate condition on hydrolysis of starch with amylase is illustrated in Figure 7.
Figure 7. The influence of substrate state over amylase activity activity 6. The determination of the degree of starch deterioration over amylase We determined the influence of the damage degree of grain starch on amylase activity in wheat flour using samples containing granular starch with different degrees of damage to the starch granule, A7, A8, A9 and A10 and amylase in wheat flour extract E1 after mode 7, using colorimetric method for the determination of amylase activity DNS reagent. Of practical results it is observed that the damage degree of the starch granule is higher, the hydrolysis reaction is easier due to amylase release from granule. This can be used as a means of correcting the baking quality of wheat. Work Mode no. 7 A 7 A 8 A 9 A 10 Starch (g) 1 1 1 1 DW (ml) 8 8 8 8 Enzyme (ml) 1 1 1 1 Incubation 15 minutes at 30 0 C Prelevation (ml) 5 5 5 5 DW (ml) 1 1 1 1 Boiling 5 minutes DW (ml) 4 4 4 4
III. CONCLUSIONS Amylase activity of wheat flour is influenced by a number of exo- and endogenous factors.the presence in the hydrolysis medium of a certain substances, determine the growth rate of starch hydrolysis reaction. This can be used practically to correct the quality of fermentative processes with low endogenous amylase by use of these substances in the production process. On the other hand, various metal ions (Ba 2+, Sr 2+, Ni 2+, K +, Li +, Al 3+, Sb 3+, Fe 3+, Cr 3+, Mn 2+, Hg 2+, NH + 4, Zn 2+, Co 2+, Cd 2+ ), anions (carbonate and dicarbonate), lowers the rate of hydrolysis of starch with amylase endogenous, so that factors can be used to improve grain quality with high amylase activity. Status of starch, the amylase concentration and the optimum conditions for amylase action are also factors that are influencing the rate of starch hydrolysis. Study of factors that alter or speed the reaction of amylase endogenous wheat flour, which involved a large number of experimental measurements was performed using colorimetric method for the determination of amylase activity using DNS reagent, modified by the authors, which was priced very well analyzes. Using this method requires, however, the performance of predeterminations for finding optimal working concentrations and volumes and some "training" regarding mode. By knowing the various factors influencing the hydrolytic activity of amylase in grains can be made practical working conditions (ph, temperature, presence or absence of effectors), which increase or inhibit the amylase activity. The fundamental objective is was to study the hydrolysis of starch amylase by analyzing the influence of exogenous and endogenous factors, frequently present in fermentative industry. The presence of cations and anions in the hydrolysis can activate or inhibit amylase activity until canceled. Calcium ion is an enzyme activator. Its concentration optimal for the hydrolysis reaction is 0.01 M.
Ions of mercury, lead, manganese and antimony totally inactivates the enzyme. The presence of anions in the hydrolysis does not change amylase activity except carbonate and sodium bicarbonate are moderate inhibitors for amylase. As such, the use of sodium bicarbonate as additional raising agent in bakery products obtained with yeast, causes a lower rate of hydrolysis of starch in dough fermentation. Status of starch (soluble or granular) has a major effect on enzymatic hydrolysis. Thus, soluble starch hydrolysis goes much faster than that of granular starch, which is influenced by the degree of damage to the starch granule, in the sense that the degree of deterioration of grain is higher, the rate of hydrolysis increases. REFERENCES [1]. Kimura, A.; Robyt, J. F. (1995) Reaction of enzymes with starch granules: kinetics and products of the reaction with glucoamylase. Carbohydr. Res. 277, 87-107. [2]. Cărăban A., Bungău S, Fodor A, Stănăşel O., (2006), Influenţa acidului ascorbic, tiaminei şi riboflavinei asupra reacţiei de hidroliză a amidonului cu amilaze endogene, utilizând interferometria laser, Revista de Chimie, 57, pp 607-609 [3]. Cǎrăban A, Stǎnǎsel O, Gavriş G, Badea G, (2008), Studies about the influence of some food lipids over the starch hydrolysis reaction using laser interferometry technics, Proceedings of Indian Conference, Impending approaches to Environmental Menace (IMAEM) 2008 TBML College, Tamil Nadu, 25-26th September, South-INDIA, pp.191-193. [4]. Hill, G.A., Macdonald, D.G. and Land, X., (1977) Alfa amylase inhibition and inactivation in barley malt during cold starch hydrolysis, Biotechologlogy Letters, 19 (11), pp. 1139-1141.
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